Notch receptors anchor a fundamental signaling pathway conserved from sea urchins to humans. Signals transduced by these receptors normally influence cell fate decisions during development, and regulate cell growth, differentiation, and death in a variety of tissue types. Aberrant Notch signals have also been implicated in vascular disease, neurodegeneration, and cancer. Notch signaling is activated when ligand binding induces metalloprotease cleavage of Notch at a site immediately external to the membrane. Proteolysis at this site triggers subsequent cleavage by gamma-secretase, which releases the intracellular portion of Notch (ICN) from the membraneand allows it to translocate to the nucleus. ICN then induces transcription of target genes by driving the assembly a Notch transcriptional activation complex (NIC) that includes a DNA-bound transcription factor called CSL and a co-activator protein of the Mastermind-like (MAML) family. The overarching goal of these studies is to uncover the factors that govern the differing sensitivities of Notch target genes to transcriptional activation in different developmental, physiologic, and pathophysiologic contexts. To address this issue, we have combined biochemical, molecular, and structural approaches to gain insight into the mechanism of transcriptional activation. In the previous period of support, we mapped the portions of human MAML1, NOTCH1, and CSL required to assemble a core Notch transcription complex (NTC) on cognate DNA, and solved the structure of this complex by X-ray crystallography. We then used insights from the structure to discover that Notch transcription complexes form dimers cooperatively on paired CSL binding sites found in a head-to-head arrangement in the promoters of certain Notch target genes. Our findings identify a new mechanistic step in Notch signaling, and lead to the hypothesis that dimerization can act as a key regulatory event in the control of Notch target gene transcription. In the next period of support, we propose to examine this hypothesis, focusing on the structure and function of NTC dimers in transcriptional activation by pursuing the following specific aims: Aim 1. Elucidate the DNA binding-site preferences for NTC monomers and dimers. Aim 2. Determine the structure of an NTC dimer on paired CSL binding sites from the promoter of a Notch target gene. Aim 3. Investigate the functional implications of Notch dimerization in models of differentiation and disease pathogenesis. Our findings will lead to new insights into the mechanism of Notch signaling, and will have broad relevance to the pathogenesis of vascular disease, neurodegeneration, and cancer.